Our major effort over the next year will be to determine the relationship of the initial Pi burst to the transition from the refractory to the non-refractory state. The Pi burst has been studied most thoroughly at 20 degrees and it appears that at this temperature the Pi burst is considerably faster than the steady-state ATPase rate. To make certain of this we will study both the Pi burst and the rate of the transition from the refractory to the non-refractory state in a single cycle of ATP hydrolysis by acto S-1 at 20 degrees. To study the Pi-burst we will use a 3-syringe stopped-flow apparatus. With this technique we should also be able to determine if the S-1 is homogeneous, i.e. whether all of it shows the initial Pi burst and follows the same kinetics or whether as postulated by Tonomura, two kinds of S-1 occur. In these experiments the S-1 will be purified using an ATP-affinity column to rule out the presence of denatured S-1. We also plan to continue our collaboration with Terrell Hill, NIAMD, quantitating our cross-bridge model to determine whether it will fit with current physiologic data.